Alpha-methylacyl CoA racemase (AMACR also known as P504S) is a metabolic enzyme that plays an important role in chiral conversion catalysis in the mitochondrial β-oxidation pathway for fatty acid catabolism and converts several (2R)-methyl-branched-chain fatty acyl-CoAs to their (S)-stereoisomers (J Lipid Res. 2000 November; 41 (11); 1890-6). AMACR has been proven to be highly expressed in prostate cancer cells and has become a novel protein biomarker for prostate cancer diagnosis (Am J Surg Pathol. 2001 November; 25 (11); 1397-404 and Cancer Res. 2000 Mar. 15; 60 (6): 1677-82). In addition, over production of AMACR would be linked to breast, colorectal, renal and other cancers in addition to prostate cancer (Am J Surg Pathol. 2002 July; 26 (7): 926-31). All of these evidences suggest that AMACR is a potent cancer biomarker and can play a synergistic role for early diagnosis of the prostate cancer. Although AMACR is a mitochondrial enzyme, there are some studies showing the possibilities of carrying out non-invasive AMACR assays directly from urine (J Urol. 2004 October; 172(4 Pt 1): 1501-3), prostate secretion (J Urol. 2004 September; 172(3) 1130-3), and blood (J Natl Cancer Inst. 2004 Jun. 2; 96 (11): 834-43) samples of the prostate cancer patients. Thus, the R&Ds of assays and sensors for detecting AMACR proteins in cancer cells or body fluids have attracted broad attention. The AMACR assay or sensor studies reported so far all relied on the use of anti-AMACR IgG antibodies as recognition elements, which are costly and have a limited shelf-life for biosensor development and production.
Aptamers are single-stranded oligonucleotides that can bind to a wide range of targets, from amino acids, drugs, proteins or even whole cells, with high affinity and specificity. In 1990, they are first selected by an in vitro selection process call SELEX (systematic evolution of ligands by exponential enrichment) (Nature. 1990 Aug. 30; 346(6287):818-22 and Science. 1990 Aug. 3; 249(4968):505-10). The process involves repeated rounds of purification from a combinatorial library of nucleic acid. Nowadays, aptamers are promising candidates for antibodies because they have several advantages that offer the possibility to overcome the limitation of antibodies, such as their rapid and easier synthesis, the capability of chemical modification, and the non-immunogenic characterization (Annu Rev Med. 2005; 56:555-83). This is an important property for application of aptamers in clinics. Although SELEX is a common practice for in vitro selection of DNA aptamers, that could still be a daunting task when the target protein is AMACR, which is a precious and costly protein. Therefore, an ordinary SELEX protocol that might consume several milligrams of protein to accomplish all SELEX rounds is not considered viable here. As illustrated in FIG. 1A, to select AMACR recognition DNA aptamers, the glass micro beads with a size of 500 μm are chosen so that the single-bead manipulation can be simply carried out by tweezers without the sophisticated instrumentation. Silane chemistry is adopted for modifying the glass bead surface with epoxide functionality for covalent immobilization of AMACR. The SELEX protocol developed in this work is rapid and consumes tiny amount of costly AMACR for accomplishing the aptamer selection rounds.